The present invention relates generally to blood coagulation, and more particularly to improved blood coagulation controls which demonstrate superior stability, and also sensitivity to variation in reagents used in coagulation tests. The coagulation control technology described herein is particularly suited to monitoring procedures for whole blood coagulation determinations for patients.
The need for stable and reliable blood coagulation controls is well documented. The continued and increased use of oral anticoagulants for treatment and management of various thrombo-embolytic conditions today, more than ever, is a driving force for their development. As is known, overdosage of anticoagulants, commonly the coumarin derivatives, can lead to serious complications, including hemorrhage from peptic ulcers, as well as other gastrointestinal complications. On the other hand, maintenance of too low a level of anticoagulant reduces or eliminates the efficacy of the prescribed treatment.
It is therefore extremely important that anticoagulant levels be reliably monitored, and, as such, a voluminous body of art has developed documenting attempts of those in the field to produce stable and reliable controls, as well as reagents, to aid in monitoring anticoagulant activity.
For example, U.S. Pat. No. 3,947,378 to Babson discloses a process for producing a control plasma deficient in Factors II, VII, IX, and X which involves treating to plasma with 20 to 22% by weight of barium sulfate at ambient temperature and then removing the adsorbent from the adsorbed plasma. The Babson patent reports that abnormal control plasmas produced by mixing the so-adsorbed plasma with normal plasma are more stable after reconstitution and give more uniform results in the activated partial thromboplastin time (APTT) procedure after storage of up to eight hours or more.
S. Zucker, M. H. Cathey, and B. West, Preparation of Quality Control Specimens for Coagulation, Amer. J. Clin. Path., June 1970, Vol. 53, pp. 924-927, reports a method for preparing lyophilized plasma specimens for use as quality controls in coagulation testing. Zucker et al. report buffering the plasma specimens with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), which was reported to provide pH and enzyme stability for prothrombin times for eight hours at 25.degree. C. An article by M. Brozovic, D. J. Howarth, L. P. van Halem Visser, and E. A. Loeliger, Stability of Freeze-Dried Plasma Prepared from Patients on Oral Anticoagulants, Journal of Clinical Pathology, 1973, Vol. 26, pp. 857-963, reported as to the suitability of freeze-dried plasmas from patients on oral anticoagulants to serve as reference material in the calibration of thromboplastins used in the control of oral anticoagulant treatment. The authors studied plasmas from several plasma pools, developed by collecting from each patient 4.5 ml of blood into 0.5 ml of a solution formed by combining 44.62 g of HEPES buffer, 38.00 g trisodium citrate (2H.sub.2 O), and 0.5 ml aprotinin TRASYLOL (1000 .mu./ml), and then adding distilled water to 1000 ml. Individual samples were centrifuged, as were the subsequently pooled samples, whereafter 1 ml of 10% sodium azide was added per liter of plasma. Samples of the prepared plasmas were then either frozen or freeze-dried. The authors reported that the freeze-dried plasmas from patients on oral anticoagulants could be used to calibrate thromboplastins provided that they are used immediately after complete reconstitution or kept at 4.degree. C. for use within four to six hours of reconstitution. The authors also reported that their plasma samples generally demonstrated varying levels and types of instability after reconstitution and storage at 4.degree. C., 22.degree. C., and 37.degree. C. It was generally reported that this instability resulted after about twenty-four hours at the lower two temperatures and after about four to six hours at the higher 37.degree. C. temperature.
U.S. Pat. No. 4,007,008 to Becker et al. describes a method for reducing enzyme activities in animal serum or plasma including the steps of raising the pH thereof to a level about that of normal serum by adding a base, and thereafter terminating the reaction or reduction of enzyme activities neutralizing the serum or plasma with an acidic medium.
Other general background can be found in U.S. Pat. No. 3,799,885 to Dennis et al. which discloses a calcium chloride test reagent buffered with HEPES buffer which is especially adapted for use in monitoring heparin therapy; in U.S. Pat. No. 4,301,028 to Bartl et al. which reports a control reagent for heparin activity determination; in U.S. Pat. No. 4,116,336 to Sorenson et al. which relates to a package containing a synthetic reference liquid for quality control and/or calibration of blood gas measuring equipment; and in P. S. Roberts, H. N. Hughes, and P. B. Fleming, The Effects of Hepes Buffer on Clotting Tests, Assay of Factors V and VIII and on the Hydrolysis of Esters by Thrombin and Thrombokinase, Thrombos, Haemostas, (Stuttg.), 1976, Vol. 35, p. 202, wherein the authors report faster clotting in the presence of 50 mM HEPES buffer.
Another aspect of the prior art control plasmas is that a majority of them, especially those commercially available, consist of or otherwise comprise primate plasma, most commonly human. These plasmas present disadvantages in that they contain unstable human factors, particularly Factors V and VIII, and also present a greater risk to the preparer or user of the controls, since they may harbor active AIDS or hepatitis viruses.
In the face of the voluminous literature and other work relating to plasmas for coagulation controls, there still remains a need for a coagulation control which exhibits superior stability with respect to one-stage prothrombin times (PT), activated partial thromboplastin times (APTT), and Factor V and VIII activity values, as well as superior sensitivity to variations in clotting test reagents employed. Certain forms of improved controls would also significantly eliminate risk of AIDS or hepatitis contraction to those who prepare and use it. For example, a coagulation product described as the CLOTrac.TM. HR control appears to be stable only for 2 hours at room temperature following reconstitution.
The applicant's invention, in its various aspects, addresses these matters.